Central chemoreception refers to the reflex regulation of breathing determined by the detection at many hindbrain sites of brain interstitial fluid (ISF) pH, which monitors the balance of three key processes, alveolar ventilation (via arterial PCO2), cerebral blood flow, and brain tissue metabolism. We hypothesize that three sites (and neurons), the retrotrapezoid nucleus (RTN: glutamatergic, Phox2b- expressing neurons), the medullary raphe (MR: serotonergic neurons), and the lateral hypothalamus (LH: orexinergic neurons) are of particular importance in sleep-related control of breathing, the upper airway and arousal. We propose that in wakefulness, the orexin neurons of the LH excite serotonergic neurons of the MR and both contribute to chemoreception via effects at the RTN. In sleep, orexinergic and medullary serotonergic neurons are less active and ventilatory chemosensitivity is reduced. In rats studied in wakefulness and sleep during both the active and quiet parts of their diurnal cycle, we manipulate these sites alone or in combination by cell specific lesions via orexin receptor-saporin conjugates, by focal dialysis of neuroactive agents, e.g., an OX1R antagonist, and by oral administration of a novel antagonist to both the OX1R and OX2R. We measure breathing as well as the electromyogram (EMG) of the major 'pump' muscle, the diaphragm, and a representative upper airway muscle, the genioglossus, in response to steady-state CO2 stimulation tests and to a newly applied dynamic CO2 test that evaluates the detection threshold at more physiological levels of CO2 elevation. We will also measure ISF pH at RTN and MR in these same conditions. This project continues our long-term focus on central chemoreception as a complex system function-not simply as a single site reflex-and focuses on chemoreceptor sites active in sleep vs wakefulness.